Novel Cell Based Assay

ABSTRACT

The present invention relates to cell-based assays involving HER2. The assays use assay cells that are transfected with cassettes containing the HER2 gene of interest and measure the effect of mutations on the activity of HER2, and on their response to inhibitors.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a Divisional of U.S. Application No.15/464,520 filed Mar. 21, 2017, now U.S. Pat. No. 10,208,357, issuedFeb. 19, 2019, the disclosure of which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the Specification. The name of the text file containingthe Sequence Listing is MDL_00066_sequence_listing_ST25. The text fileis 9,203 bytes was created on Mar. 10, 2017.

FIELD OF THE INVENTION

The present invention generally relates to cell-based assays involvingHER2 and HER2 protein. These cell-based assays are used to measure theeffect of mutations on the activity of HER2 protein and on theirresponses to inhibitors.

BACKGROUND OF THE INVENTION

HER2 protein is a member of the human epidermal growth factor receptorfamily. There are four members of this family which are plasma membranebound receptor tyrosine kinases. Breast cancer is the most prevalentcancer in women, and about 25% of cases show HER2 gene amplification.Breast cancers with HER2 gene amplification or HER2 proteinoverexpression are typically referred to as HER2-positive in pathologyreports. HER2-positive breast cancers tend to grow faster and are morelikely to spread and recur compared to HER2-negative breast cancers.HER2 amplification is a major therapeutic target in breast cancer. Thereare medicines specifically for HER2-positive breast cancers. Lapatinib,which is an orally active drug approved for breast cancer treatment, isa dual tyrosine kinase inhibitor which targets both HER1 and HER2tyrosine kinase activity. Two therapeutic antibodies, includingTrastuzumab and Pertuzumab, targeting HER2 receptor are also approved bythe FDA for breast cancer patients.

Cancer evolution and progression are driven by a sequence of somaticgenetic and nongenetic alterations resulting in more favorable tumorcell growth and survival. Cancer genetic evolution is subject tointrinsic influences such as the tumor microenvironment, as well asextrinsic pressures such as drug therapy. The clinical pattern ofacquired resistance may, in many circumstances, represent outgrowth ofresistant clones, which may have originally been present in the cancerat low frequency as a result of intratumoral genetic heterogeneity, butgrow out under the selective pressure of targeted therapy.

Advances in high-throughput sequencing technologies are beginning toestablish a molecular taxonomy for a spectrum of human disease and havefacilitated a move toward precision medicine. With regard to oncology,defining the mutational landscape of a patient’s tumor will lead to moreprecise treatment and management of individuals with cancer. In additionto the potential for identifying ‘actionable’ therapeutic targets incancer patients, the clinical sequencing may also shed light on acquiredresistance mechanisms developed against targeted therapies. Althoughuncovering the DNA sequences of tumors becomes possible with theadvances in next generation of sequencing (“NGS”), this technology doesnot provide any functional information of the identified mutations.Therefore, there is a need to develop a novel method to study thefunctional consequence of mutation(s) on the target gene activity andits response toward the drug treatment. This functional information willprovide additional valuable guidelines for the physician to choose themost appropriate treatment based on the mutational landscape of apatient’s tumor.

Accordingly, there is a continuing need to develop an improved clinicaltest using a cell-based assay that measures HER2 activity useful inassessing the effect of somatic HER2 gene mutations in the respectiveprotein activity, as well as determining the sensitivity of HER2variants to inhibitors.

SUMMARY OF THE INVENTION

In one aspect the present invention concerns a method of determiningwhether an HER2 variant is sensitive to treatment with an HER2 inhibitorin a cell, comprising the steps of:

-   a) preparing a cDNA encoding said HER2 variant;-   b) preparing an expression cassette containing the HER2 variant    cDNA;-   c) transfecting said prepared expression cassette containing said    HER2 variant cDNA in an assay cell having a JNK reporter construct    comprising a reporter gene cDNA linked to at least one AP-1 binding    site, and said cell is capable of expressing HER3;-   d) exposing said transfected cell to a HER3 ligand, wherein HER3    complexes with said HER2 variant to form a dimer which thereby    activates the JNK reporter construct and generates a signal;-   e) exposing said transfected cell with an HER2 inhibitor; and-   f) determining whether said HER2 variant is sensitive to treatment    with said HER2 inhibitor by measuring a change in signal.

In another aspect the present invention concerns a method of determiningthe activity of a HER2 variant, comprising the steps of:

-   a) preparing a cDNA encoding said HER2 variant;-   b) preparing an expression cassette containing the HER2 variant    cDNA;-   c) transfecting said prepared expression cassette containing said    HER2 variant cDNA in a cell having a JNK reporter construct    comprising a reporter gene cDNA linked to at least one AP-1 binding    site, and said cell is capable of expressing HER3;-   d) exposing said transfected cell to a HER3 ligand, wherein said    HER3 complexes with said HER2 variant to form a dimer which thereby    activates the JNK reporter construct and generates a signal;-   e) determining the signal activity, wherein a change in signal    activity relative to wild type HER2 is indicative of a change in    activity of said HER2 variant.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the PCR mediated overlapping extension to constructpatient HER2 gene. Two rounds of PCR were performed. The first round ofPCR included two independent PCR as illustrated as PCR#1 and PCR#2. HER2expression plasmid was used as DNA template. Second round of PCR wasperformed after ExoSAP-IT® treatment. Mixture of the PCR products fromfirst round of PCR was used as DNA template. The mutation wasincorporated into the final linear expression cassette, which containedCMV promoter and BGH terminator, as illustrated in the figure.

FIG. 2 depicts the luciferase reporter activity of HEK 293 cellstransfected with active luciferase reporter using reverse and forwardtransfection methods.

FIG. 3 depicts the immunoblot result using antibodies against HER1, HER2and HER3 receptors. HEK 293 cells were transfected with nothing orexpression plasmid of HER1, HER2 or HER3.

FIG. 4 depicts the result of genomic PCR screening of HER2 CRISPRknockout cells. A) HEK 293 cells were transiently transfected with theHER2 CRISPR constructs. B) Single cell clones of cells transfected withthe HER2 CRISPR constructs.

FIG. 5 depicts the immunoblot result of HER2 knockout cell clone usingantibody against HER2 receptor. Antibody against GAPDH was used aloading control.

FIG. 6 depicts the immunoblot result of HER1 and HER2 double knockoutclones using antibodies against HER1 and HER2 receptors. Antibodyagainst GAPDH was used a loading control.

FIG. 7 depicts the luciferase reporter activity of HEK 293 cellstransiently transfected with A) ERK-Luc, B) FRK-Luc, C) JNK-Luc and D)NFkB-Luc in the presence or absence of HER2 expression plasmid.

FIG. 8 depicts the luciferase reporter activity of HEK 293 cellstransiently transfected with A) ERK-Luc, B) FRK-Luc, C) JNK-Luc and D)NFkB-Luc in the presence or absence of HER2 expression plasmid. The HER2transfected cells were also treated with 10 nM of Lapatinib for 4 hours.

FIG. 9 depicts the luciferase reporter activity of HEK 293 cellstransiently transfected with NFkB-Luc reporter in the absence orpresence of wild-type (WT), constitutively active (CA) or kinase-dead(KD) of HER2 expression plasmid.

FIG. 10 depicts the luciferase reporter activity of HEK 293 cellstransiently transfected with NFkB-Luc reporter in the absence orpresence of HER2 expression plasmid. The transfected cells were alsotreated with the HER2 inhibitors including Lapatinib, Trastuzumab andPertuzumab.

FIG. 11 depicts the luciferase reporter activity of HEK 293 cellstransiently transfected with A) ERK-Luc, B) JNK-Luc, C) NFkB-Luc in thepresence of HER1, HER2 and HER3 expression plasmids. The transfectedcells were treated with HER1 ligand (EGF) or HER3 ligand (NRG-1).

FIG. 12 depicts the luciferase reporter activity of HEK 293 cellstransiently transfected with JNK-Luc reporter in the presence of HER1and wild-type (WT) or kinase-dead (KD) HER2 expression plasmids. Thetransfected cells were treated with HER1 ligand (EGF).

FIG. 13 depicts the luciferase reporter activity of HEK 293 cellstransiently transfected with A) ERK-Luc and B) JNK- Luc reporters in thepresence of HER3 and wild-type (WT) or kinase-dead (KD) HER2 expressionplasmids. The transfected cells were treated with HER3 ligand (NRG-1).

FIG. 14 depicts the luciferase reporter activity of HEK 293 cellstransiently transfected with JNK-Luc reporter in the absence or presenceof HER2 and HER3 expression plasmids. The transfected cells were treatedwith a serial dilution of HER3 ligand (NRG-1).

FIG. 15 depicts the luciferase reporter activity of HEK 293 cellstransiently transfected with 6X JNK- Luc reporter in the absence orpresence of HER2 and HER3 expression plasmids. The transfected cellswere treated with HER3 ligand (NRG-1).

FIG. 16 depicts the luciferase reporter activity of double knockoutclone #27 (HEK293 DKO#27) transiently transfected with 6X JNK-Lucreporter in presence of HER2 and HER3 expression plasmids. Thetransfected cells were treated with a serial dilution of HER3 ligand(NRG-1).

FIG. 17 depicts the luciferase reporter activity of double knockoutclone #27 (HEK293 DKO#27) transiently transfected with 6X JNK-Lucreporter in presence of HER2 and HER3 expression plasmids. Thetransfected cells were treated with HER3 ligand (NRG-1) in the presenceor absence of inhibitors of HER2.

FIG. 18 depicts the luciferase reporter activity of double knockoutclone #27 (HEK293 DKO#27) transiently transfected with 6X JNK-Lucreporter in presence of HER3 and wild-type (WT), constitutively active(CA) or kinase-dead (KD) HER2 expression plasmids. The transfected cellswere treated with a serial dilution of HER3 ligand (NRG-1).

FIG. 19 depicts the luciferase reporter activity of single cell clonesstably integrated with 6X JNK-Luc. These single cell clones weretransiently transfected with HER2 and HER3 expression plasmids followedby treatment of NRG1. A) Relative luciferase activity was shown. B) Foldof activation upon the treatment of NRG1 was calculated.

FIG. 20 depicts the luciferase reporter activity of single cell clones#4, 11, 28 and 33, which were stably integrated with 6X JNK-Luc. Thesesingle cell clones were transiently transfected with HER2 and HER3expression plasmids followed by treatment of NRG1. A) Relativeluciferase activity was shown. B) Fold of activation upon the treatmentof NRG1 was calculated.

FIG. 21 depicts the luciferase reporter activity of single cell clone#28 transiently transfected with the HER3 expression plasmid and thelinear expression cassette of HER2. The transfected cells were treatedwith a serial dilution of HER3 ligand (NRG-1).

FIG. 22 depicts the luciferase reporter activity of single cell clone#28 transiently transfected with the HER3 expression plasmid and thelinear expression cassette of wild-type (WT), constitutively active (CA)or kinase-dead (KD) of HER2. The transfected cells were treated with aserial dilution of HER3 ligand (NRG-1).

FIG. 23 depicts the luciferase reporter activity of single cell clone#28 transiently transfected with the HER3 expression plasmid and thelinear expression cassette of wild-type (WT) HER2. The transfected cellswere treated with NRG1 and HER2 inhibitors including Lapatinib,Trastuzumab and Pertuzumab.

FIG. 24 depicts the luciferase reporter activity of eight single cellclones stably integrated with 6X JNK-Luc and HER3 expression plasmid.These single cell clones were transiently transfected with HER2expression plasmid followed by treatment of NRG1. A) Relative luciferaseactivity was shown. B) Fold of activation upon the treatment of NRG1 wascalculated.

FIG. 25 depicts the luciferase reporter activity of the HER2 reportercell transiently transfected with the wild-type (WT), constitutivelyactive (CA) or kinase-dead (KD) of HER2 expression plasmid in the384-well plate. The transfected cells were treated with HER3 ligand(NRG-1).

FIG. 26 depicts the luciferase reporter activity of the HER2 reportercell transiently transfected with the linear expression cassette of HER2in the 384-well plate. The transfected cells were treated with a serialdilution of HER3 ligand (NRG-1) for A) 4 hours, B) 6 hours and C) 24hours.

FIG. 27 A) Schematic representation of the “all-in-one” stimulationmethod. The NRG-1 was added to the cells together with the DNAtransfection mix, i.e,, at the same time. The transfected/treated cellswere incubated for 24 hours. The luciferase activity was measured. B)depicts the luciferase reporter activity of the HER2 reporter celltransiently transfected with the linear expression cassette of HER2 inthe 384-well plate. The transfected cells were treated with a serialdilution of HER3 ligand (NRG-1) using the “all-in-one” method.

FIG. 28 depicts the receptor activity of wild-type HER2 receptor and theinhibitory activities of Lapatinib, Trastuzumab and Pertuzumab using theHER2 reporter cells transiently transfected with 50 ng of linearexpression cassette of wild-type HER2. The transfected cells weretreated with different concentrations of NRG-1 and HER2 inhibitors using“all-in-one” method. A) The luciferase reporter activity was measuredand illustrated. B) The percentage activity of estrogen receptor wascalculated and summarized in the table.

FIG. 29 depicts the receptor activity of L755S HER2 receptor and theinhibitory activities of Lapatinib, Trastuzumab and Pertuzumab using theHER2 reporter cells transiently transfected with 50 ng of linearexpression cassette of L755S HER2. The transfected cells were treatedwith different concentrations of NRG-1 and HER2 inhibitors using the“all-in-one” method. A) The luciferase reporter activity was measuredand illustrated. B) The percentage activity of estrogen receptor wascalculated and summarized in the table.

FIG. 30 depicts the receptor activity of P780ins HER2 receptor and theinhibitory activities of Lapatinib, Trastuzumab and Pertuzumab using theHER2 reporter cells transiently transfected with 50 ng of linearexpression cassette of P780ins HER2. The transfected cells were treatedwith different concentrations of NRG-1 and HER2 inhibitors using the“all-in-one” method. A) The luciferase reporter activity was measuredand illustrated. B) The percentage activity of estrogen receptor wascalculated and summarized in the table.

FIG. 31 depicts the receptor activity of T862A HER2 receptor and theinhibitory activities of Lapatinib, Trastuzumab and Pertuzumab using theHER2 reporter cells transiently transfected with 50 ng of linearexpression cassette of T862A HER2. The transfected cells were treatedwith different concentrations of NRG-1 and HER2 inhibitors using the“all-in-one” method. A) The luciferase reporter activity was measuredand illustrated. B) The percentage activity of estrogen receptor wascalculated and summarized in the table.

ETAILED DESCRIPTION OF THE INVENTION

The present invention can be better understood from the followingdescription of preferred embodiments, taken in conjunction with theaccompanying drawings. It should be apparent to those skilled in the artthat the described embodiments of the present invention provided hereinare merely exemplary and illustrative and not limiting. Numerousembodiments or modifications thereof are contemplated as falling withinthe scope of the present invention and equivalents thereto. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, mitigating or inhibiting theprogress of the disorder or condition to which such term applies, or oneor more symptoms of such disorder or condition. As used herein and inthe appended claims, the singular forms “a”, “an”, and “the” includeplural reference unless the context clearly dictates otherwise.

Definitions

Various terms used in this specification shall have the definitions setout herein. All derivatives, inflections and conjugations or othergrammatical forms of a specific term are intended to be included in therecited definition.

As used herein, the term “A,” “T,” “C”, and “G” refer to adenine,thymine, cytosine, and guanine as a nucleotide base, respectively.

As used herein, the term “AP-1” refers to activator protein 1 which is atranscription factor activated by JNK proteins which has the sequenceTGAGTCAG . AP-1 is capable of modulating gene expression in response tobinding of certain ligands to HER cell surface receptors. Receptoroccupancy triggers a signal transduction cascade to the nucleus. In thispathway or cascade, transcription factors such as AP-1 execute long termresponses to the extracellular factors by modulating gene expression.

As used herein the term “HER” is a receptor protein tyrosine kinasewhich belongs to the HER receptor family and includes EGFR (ErbB1,HER1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) receptors. The HERreceptor will generally comprise an extracellular domain, which may bindan HER ligand and/or dimerize with another HER receptor molecule; alipophilic transmembrane domain; a conserved intracellular tyrosinekinase domain; and a carboxyl-terminal signaling domain harboringseveral tyrosine residues which can be phosphorylated.

As used herein, the term “HER2” (also known as receptor tyrosine-proteinkinase erbB-2 or ERBB2) refers to human epidermal growth factor receptor2 which is encoded by HER2 having reference number NM_004448 in theNational Center for Biotechnology Information (“NCBI”) database.

As used herein, the term “HER1” refers to human epidermal growth factorreceptor 2 which is encoded by HER1 having NCBI reference numberNM_005228.

As used herein, the term “HER3” refers to. human epidermal growth factorreceptor 3 which is encoded by HER3 having NCBI reference numberNM_001982.

As used herein the term “wild type” or “WT” means the typical form of anorganism, strain, gene or characteristic as it occurs in nature asdistinguished from mutant, variant, or modified forms.

As used herein the term “variant” means the exhibition of qualities thathave a pattern that deviates from what occurs in nature or is distinctfrom the predominant form that occurs in nature.

As used herein, the term “vehicle” refers to the solvent of a compound.

As used herein, the term “CRISPR” refers to Clustered regularlyinterspaced short palindromic repeats, which are sequences used byCRISPR associated proteins (Cas) for the purpose of recognizing andcutting genetic elements. CRISPR/Cas9 uses sgRNA as a recognitionsequence for identifying where the Cas9 will bind and cut the geneticelement.

As used herein, the term “cancer” refers to a malignant neoplasticdisease. Most cancers are characterized by hyperproliferation of a cellpopulation.

As referred to herein, the term “assay cell” refers to a cell which istransfected for use in the assays of the invention.

As used herein, the term “tumor cell” or “cancer cell” refers to amalignant neoplastic cell.

As used herein, the term “luciferase activity” refers to the use of aluciferase protein or reporter to assess the amount of luciferase lightemission. The activity is measured by addition of a substrate that bindsto the luciferase protein and emits a light signal that can be measuredusing a luminometer.

As used herein, the term “promoter” refers to a region of the DNA thatregulates the transcription of a particular gene.

As used herein, “expression” or “expressed” refers to the processes bywhich a polynucleotide is transcribed from a DNA template (such as intoand mRNA or other RNA transcript) and further processed or translatedinto peptides, polypeptides, or proteins. Transcripts and encodedpolypeptides may be collectively referred to as “gene product.” If thepolynucleotide is derived from genomic DNA, expression may includedifferential splicing of the mRNA in a eukaryotic cell leading todifferent forms of peptides or protein products.

As used herein, the term “construct” refers to a plasmid orpolynucleotide, e.g., a linear cassette, containing cDNA to encode for agiven protein. Constructs typically have the necessary components toexpress the desired protein encoded by the cDNA.

As used herein, the terms “stable expression” or “stably expressing”refer to the a cell line or group of cells that express a given proteinfor a period greater than 1 week, normally resulting in permanentexpression of that protein over months.

As used herein, the term “stable cell” or “stable cell system” refers tothe generation of cells using a selection method that specificallystably express a given protein. “Stable cell clone” is derived from asingle cell with stable expression of a given protein.

As used herein, the term “transfection” refers to the process ofintroducing a polynucleotide into a cell, and more specifically into theinterior of a membrane-enclosed space of a target cell(s), such as thecytosol of a cell, the nucleus of a cell, an interior space of amitochondria, endoplasmic reticulum (ER), and the like. Transfection canbe accomplished by any means known to those of skill in the art, forexample as taught by Sambrook et al. Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, New York (2001), thecontents of which are incorporated by reference herein. Examples oftransfection techniques include, but are not limited to: heat shock,calcium phosphate transfection, PEI transfection, electroporation,lipofection, transfection reagent(s), viral-mediated transfer, and thelike, or combinations thereof.

As used herein, the term “transiently transfected” refers to a cell thathas been subject to a process of introducing a polynucleotide into acell resulting in expression of a protein over a period from 12 hours to7 days.

As used herein, the term “HER2 inhibitor” refers to a compound whichtargets and binds to HER2 or otherwise interacts directly or indirectlywith HER2, which interferes with HER activation or function resulting inprevention or reduction of HER activity.

As used herein the term “HER3 ligand” refers to a compound, e.g., apolypeptide, which binds to and/or activates the HER3 receptor.

As used herein the term “HER2 activity” refers to the ability of HER2 tointeract with other HER receptors to form heterodimers to inducedownstream signaling activity.

As used herein the term “reporter” means a protein that when expressedin a cell is capable of producing a detectable signal.

As used herein the term “reporter gene” refers to a polynucleotide thatencodes a reporter.

As used herein the term “JNK reporter construct” refers to apolynucleotide that contains cDNA encoding a reporter protein, typicallyan enzyme such as luciferase, and at least one copy of AP-1. An exampleof a JNK reporter construct is SEQ ID NO: 31.

As used herein the term “HER activation” refers to activation, orphosphorylation, of any one or more HER receptors.

As used herein the term “JNK protein” refers to a member of the JNKfamily of kinases, including but not limited to Jun N-terminal kinase 1(“JNK1”), Jun N-terminal kinase 2 (“JNK2”) and Jun-N-terminal kinase 3(“JNK3”), their isoforms and other members of the JNK family of proteinsthat phosphorylate the amino terminal (N-terminal) portion of the Junsubunit of AP-1.

As used herein the term “dead” form of HER2 or HER2 refers to HER2receptor containing a missense mutation at amino acid position 753. Thismutation changes lysine to methionine, which results in inactivation ofkinase activity of HER2 receptor

In a typical situation a cell converts an extracellular signal orstimulus into a response, typically involving ordered sequences ofbiochemical reactions inside the cell. The phosphorylation of proteinsplays a key role in the transduction of extracellular signals into thecell. Certain cell signaling pathways involve mitogen activated proteinkinases, the so-called MAP (mitogen activated protein) kinases or MAPK.One of the MAPK-dependent pathways enables the transmission of signalsfrom extracellular signals, such as neuregulin 1 or NRG1 which binds toa corresponding receptor in the cell membrane, i.e., the HER2/HER3dimer, which sends the signal on to the cell nucleus via intermediarykinases and kinase targets (e.g., the MAPK pathway). Latter proteins inthis pathway are JNK proteins which ultimately govern expression ofgenes that control vital cell functions such as proliferation, growth,motility and survival. JNK proteins are responsible for thephosphorylation of specific sites (Serine 63 and Serine 73) on the aminoterminal portion of c-Jun. Phosphorylation of these sites potentiatesthe ability of AP-1 to activate gene expression.

The epidermal growth factor receptor family includes four transmembranetyrosine kinase receptors named HER1, HER2, HER3 and HER4. HER receptorsshare a highly conserved extracellular domain, a transmembrane junction,and an intracellular ATP-binding kinase domain. Several ligands havebeen described to bind with different specificities to the extracellulardomain of the HER receptors. Upon ligand binding, HER receptors formdimers that, following transphosphorylation of their kinase domains,recruit adaptor molecules responsible for the initiation of severalsignaling pathways involved in cell proliferation and survival. Althoughdifferent dimer combinations of the four receptors are possible, theHER1/HER2 and HER2/HER3 heterodimers are considered the most potent andoncogenic combination. Therefore, it is desirable to characterize theexpression of HER1, HER2 and HER3 in assay cells, e.g., transfectedHEK293, MCF7, HeLa cells, and the like. If there are detectable amountsof these receptors in the assay cells in one embodiment the endogenousgenes can be knocked out to eliminate potential interference with theassay.

CRISPR/Cas9 can be used to generate knock-out (“KO”) cells byco-expressing a gRNA specific to the gene to be targeted and theendonuclease Cas9. An assay cell with two genes knocked out is referredto as a double knock-out or “DKO.” The genomic target can be any 20nucleotide DNA sequence, provided it meets two conditions:

-   1. The sequence is unique compared to the rest of the genome.-   2. The target is present immediately upstream of a Protospacer    Adjacent Motif (“PAM”).

The PAM sequence is necessary for the target in order to providespecificity. Cas9 is the endonuclease from the species of Streptococcuspyogenes. Once expressed, the Cas9 protein and the gRNA form ariboprotein complex through interactions between the gRNA “scaffold”domain and surface-exposed positively-charged grooves on Cas9. Cas9undergoes a conformational change upon gRNA binding that shifts themolecule from an inactive, non-DNA binding conformation, into an activeDNA-binding conformation. The Cas9-gRNA complex will bind any genomicsequence with a PAM, but the extent to which the gRNA spacer matches thetarget DNA determines whether Cas9 will cut. Once the Cas9-gRNA complexbinds a putative DNA target, a “seed” sequence at the 3′ end of the gRNAtargeting sequence begins to anneal to the target DNA. If the seed andtarget DNA sequences match, the gRNA will continue to anneal to thetarget DNA in a 3′ to 5′ direction. Cas9 will only cleave the target ifsufficient homology exists between the gRNA spacer and target sequences.The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH.Cas9 undergoes a second conformational change upon target binding thatpositions the nuclease domains to cleave opposite strands of the targetDNA. The end result of Cas9-mediated DNA cleavage is a double strandbreak (“DSB”) within the target DNA (3-4 nucleotides upstream of the PAMsequence). The resulting DSB is then repaired by one of two generalrepair pathways:

-   1: The efficient but error-prone Non-Homologous End Joining (“NHEJ”)    pathway-   2: The less efficient but high-fidelity Homology Directed Repair    (“HDR”) pathway

The NHEJ repair pathway is the most active repair mechanism, capable ofrapidly repairing DSBs, but frequently results in small nucleotideinsertions or deletions (“InDels’) at the DSB site. In most cases, NHEJgives rise to small InDels in the target DNA which result in in-frameamino acid deletions, insertions, or frameshift mutations leading topremature stop codons within the open reading frame (“ORF”) of thetargeted gene. Ideally, the end result is a loss-of-function mutationwithin the targeted gene; however, the “strength” of the knock-outphenotype for a given mutant cell is ultimately determined by the amountof residual gene function.

The HER family proteins are type I transmembrane growth factor receptorsthat function to activate intracellular signaling pathways in responseto extracellular signals. Their structure consists of an extracellularligand binding domain, a transmembrane domain, and an intracellulartyrosine kinase domain. The extracellular domain of HER proteins canexist in a closed inhibited or an open active conformation. Ligandbinding causes a conformational change in their extracellular domainthat induces the active conformation and promotes their dimerization andconsequent transphosphorylation. Partner selection appears to be a keydeterminant of signaling activity among HER proteins and their signalingfunctions follow a distinct hierarchical order favoring heterodimersover homodimers. HER2 has the strongest catalytic kinase activity andHER2-containing heterodimers have the strongest signaling functions.Unlike the other members of the family, HER2 lacks ligand bindingactivity and its signaling function is engaged by its ligand-boundheterodimeric partners.

Unlike the other members of the family, the extracellular domain of HER2does not pivot between active and inactive conformations andconstitutively exists in an activated conformation. Consistent with itsconstitutively active conformation, HER2 lacks ligand binding activityand its signaling function is engaged by its ligand-bound heterodimericpartners. On the other hand HER3, unlike the other members, lacks ATPbinding within its catalytic domain and is catalytically inactive.Consistent with this, the signaling functions of HER3 are mediatedentirely through the kinase activity of its heterodimeric partners.Although individually they are incomplete signaling molecules, a largebody of evidence not only establishes HER2 and HER3 as obligate partnersbut their complex forms the most active signaling heterodimer of thefamily and essential for many biologic and developmental processes. HER1contains both ligand binding activity and its cytosolic catalytic kinasedomain. HER1 can form homodimer or/and heterodimer to engage itssignaling function.

In general, there are two major types of transfection, forward andreverse. The most routinely employed transfection protocol where cellsare seeded a day prior to transfection is referred to as “forwardtransfection”. Forward transfection methods work well for most adherentcell types that are seeded a day prior to transfection in order toachieve an actively dividing cell population at the time oftransfection. A “reverse transfection” protocol where freshly passagedcells are added to transfection complexes has the advantage of reducinghands-on time for the end user. In this scenario, cells are not adheredto the plate surface by the time they interact with the transfectioncomplexes.

The present invention relates to HER2 variants sensitivity to treatment,HER2 variants sensitivity to inhibitors and whether compounds inhibitHER2 activity in a cell. In one aspect, the present invention provideshighly sensitive methods for determining whether a HER2 variant issensitive to treatment using a HER2 inhibitor.

Methods include providing a cell system containing cDNA constructs withsignaling capability to measure the activity of HER2 variants and thevariants response to treatment with inhibitors. The HER2 variants mayinclude one or more mutations in the respective gene. Identification ofthe HER2 variants may be obtained from sequencing of a biological sampleor produced using, e.g., Next Generation Sequencing (NGS).

The assay cells of the invention are transfected with a reporter genecontaining one or more AP-1 binding sites. The assay cells can be eithertransiently or stably transfected with the JNK reporter construct. In aparticular embodiment the assay cells contain a stably integrated JNKreporter construct. When HER2/HER3 dimer is activated by a HER3 ligand,a biochemical cascade ensues which involves various kinases whichresults in the reporter gene being expressed and production of a signal.In some embodiments in order to produce a signal a substrate must beprovided for the reporter. The signal can be, for example, a lightsignal. In some embodiments the reporter is an enzyme which can be anyprotein produced from any gene that exhibits enzymatic activity anddegrades a substrate to produce a light or luminescence signal. Thelight signal can be measured using a luminometer. In certainembodiments, the light is measured by fluorescence signaling systemssuch as Fluorescence Resonance Energy Transfer (FRET).

Examples of reporter enzymes include luciferase, alkaline phosphatase,chloramphenicol transferase, β-galactosidase, β-glucuronidase,carboxylesterase, lipases, phospholipases, sulphatases, ureasespeptidases, proteases and the like.. In a particular embodiment thereporter is luciferase, for example, firefly luciferase, Renillaluciferase, and the like. In one aspect, the present invention providesa method to determine HER2 gene activity using a cell based reporterassay. Ligand-dependent nuclear transactivation involves activation ofthe JNK pathway. In the assays of the invention the stable assay cellscontain a reporter construct which comprises a reporter gene containingAP-1 binding sites. Binding of a HER3 ligand to the HER2/HER3 dimerresults in a signal. In one embodiment HER3 ligand binding inducesexpression of the reporter which leads to generation of light emissionwhen a substrate is added, and therefore allows for indirect measurementof HER2 activity. Typical substrates for luciferase include D-luciferinand salts thereof.

The present assay can be used in personalized medicine. When genomeinformation is obtained relating to the HER2 or HER2 sequences, oneskilled in the art can conveniently prepare a cDNA based on the HER2gene sequence information. The generated cDNA therefore contains aunique cDNA for that individual because it contains a gene sequence ofthe specific gene of that individual. The HER2 construct containingeither the variant or WT HER2 can be transfected into the assay cell viaeither a plasmid or linear expression cassette. In certain embodiments,the cDNA encoding the HER2 variant may be transiently transfected intothe cell. One advantage of a preferred assay of the invention is totransfect the generated HER2 cDNA into an assay cell with a linearcassette. Compressing or shortening the time required to perform theassays of the invention can be important when performing the assays in aclinical laboratory. By use of linear cassette transfection the timerequired to obtain and report results to a physician can besubstantially shortened relative to prior art methods using plasmids.For example performing the assays of the invention (i.e., steps (a)through (f) of the inhibitor sensitivity assays, and steps (a) through(e) of the activity assays) can take 32 hours or less, typically 30hours or less, preferably 28 hours or less.

Normally, transfection with the linear cassette takes about 4 to 24hours, in some embodiments about 5 to 20 hours, in another embodimentabout 6 to 12 hours. In one embodiment, once the assay cells aretransfected with the HER2 cassette, the assay cells are cultured orincubated under conditions suitable for adequate cell growth andexpression of the proteins of interest; in one embodiment the conditionsinclude a culture time of 24 to 48 hours typically at about 37° C. Aftertransfection, the assay cells are exposed to a HER3 ligand for about 2to 6 hours. If a HER2 inhibitor is used in the assay, the assay cellsare exposed to said inhibitor for about 2 to 6 hours. In a certainembodiment, contacting or exposure with inhibitor is performed afterincubating the assay cells with a HER ligand inducing HER dimerization.

The HER ligand can be any compound that binds to or interacts with HER3and activates the receptor. HER ligands can be polypeptides. The HERligand can be an antibody or fragment thereof, alpha, beta and gammaheregulins; neuregulin-1 (“NRG1”), neuregulion-2, neuregulin-3,neuregulin-4, and the like.

The ligand and inhibitor in one embodiment are added to the assay cellsat about the same time, i.e., simultaneously. In another embodiment, ithas been surprisingly discovered that ligand can be added at about thesame time as transfection with the HER2 cassette, i.e., performing stepsc) and d) simultaneously (termed herein as the “all-in-one” method), andstill result in a good dose response to the respective ligand.

Transfection with the linear expression cassette can be a “forwardtransfection” or a “reverse transfection.” Forward transfection is wherecells are seeded a day prior to transfection in order to achieve anactively dividing cell population adhered to a vessel surface at thetime of transfection. Reverse transfection is where freshly passagedcells are added to transfection complexes. Reverse transfection has theadvantage of reducing the time for performing assays; however, becausethe cells are not adhered to the surface of the assay vessel (e.g.,surface of the wells of the assay plate) and are not in a robust growthphase, reverse transfection can be unsuitable for certain assays. It hasbeen discovered that for the assays of the invention reversetransfection results in satisfactory signal production, in particularluciferase production, in the assay cells.

In certain embodiments, the assay cell is transfected with a cDNAcontaining HER2 gene of interest, typically a variant. The cDNA can beconveniently prepared using standard methodologies known to one skilledin the art. In certain embodiments, the cDNA can encode HER2 wild type.In certain embodiments the cDNA can encode a HER2 variant. In furtherembodiments, the variants can contain one or more mutations differentfrom the HER2 wild type. The HER2 construct containing either thevariant or WT HER2 can be either in a plasmid or linear expressioncassette format. In certain embodiments, the HER2 variant contains onemutation. In certain embodiments, the specific variant may contain twomutations. In certain embodiments, the variant may contain threemutations. In certain embodiments the variant may contain four or moremutations.

In some embodiments, the HER2 variant contains a missense mutation,insertion, or deletion. Examples of missense mutations in HER2 areT862A, L755S, and the like. An example of an insertion for HER2 isP780ins.

In addition to the HER2 cDNA encoding a variant, the expressioncassette, preferably linear, also comprises other components necessaryor desirable for effective expression. These components may varydepending on the particular assay cell chosen. Such other componentstypically include a promoter and terminator. Promoters include thecytomegalovirus (CMV) promoter, the SV40 promoter, elongation factor(EF)-1 promoter and the like. Typical terminators are SV40, hGH, BGH,and rbGlob. In addition a polyadenylation or poly(A) signal sequence istypically included.. The assay cells can be either stably or transientlytransfected with the HER2 expression cassette, but it typically istransiently transfected.

In some embodiments the assays cells are stably transfected with JNKreporter constructs. Stable transfection has the advantage of passingthe DNA to the progeny of the cells. Stable transfection typicallyincorporates the transfected DNA into the genome of the assay cell, butit is possible that transfected DNA can be stable even though notincorporated into the genome. In some embodiments the assay cells aretransfected with JNK reporter constructs that have multiple copies ofAP-1 binding sites, e.g., 2, 3, 4, 5, 6, 9, 12 or more copies. In oneembodiment the reporter construct has 6 or more AP-1 binding sites, in aparticular embodiment the signal expression construct has 6 AP-1 bindingsites. Usually multiple copies of AP-1 result in enhanced generation ofsignal. The introduced reporter construct is preferably integrated intothe host genome and retained in the cells even after assay cellsreplicate. The JNK reporter constructs also comprises other componentsnecessary or desirable for effective stable transfection, similar to theHER2 and HER3 constructs. These components may vary depending on theparticular host or parent cell chosen. In addition to components such asa promoter, terminator and polyA sequence, such other components caninclude, for example, a marker gene for selecting and identifying cellscontaining the reporter constructs integrated into the host genome. Suchmarker gene can be, for example, genes that encode a fluorescent proteinor encode antibiotic resistance such as resistance to hygomycin B,neomycin, puromycin and the like. The marker gene can be part of thesignal expression construct or can be part of a separate constructco-transfected with the signal expression construct.

The assay cells of the invention are capable of expressing HER3. Theexpression of HER3 is important in order for the expressed HER3 todimerize with HER2 expressed for the transfected HER2 cDNA. If theparticular assay cell chosen expresses sufficient endogenous HER3, thenendogenously expressed HER3 may be sufficient. However, if endogenousHER3 expression is low or absent, then it may be desirable to transfectthe assay cell with an additional construct containing HER3 cDNA. Such aHER3 construct will comprises other components necessary or desirablefor effective transfection. These components may vary depending on theparticular assay cell chosen. Such other components typically include apromoter, terminator and poly A sequences as described above. The HER3construct can contain multiple copies of the HER3 gene. The assay cellcan be either stably or transiently transfected with the HER3 expressionconstruct, but it is preferably stably transfected.

The assay cells of the invention can be transfected with the constructsdescribed herein individually via a plasmid or linear expressioncassette or the desired genes can be part of the same plasmid or linearexpression cassette. For example, in one embodiment both the JNKreporter construct and the HER3 construct are contained in the sameplasmid or linear expression construct. In another embodiment both theJNK reporter and HER3 are in two separated plasmids and they are bothstably integrated into the genome.

In another aspect, the present invention provides a method to determinewhether a particular HER2 variant is sensitive to treatment with a HER2inhibitor in a cell. The method involves preparing a cDNA containing aHER2 variant of interest followed by transfecting the cDNA into an assaycell. In certain embodiments, the transfected cells are then exposed toan inhibitor. HER 2 inhibitors include, without limitation, HERantibodies and antibody fragments, small molecule HER2 antagonists, HER2tyrosine kinases inhibitors, and antisense molecules. In one embodiment,the HER2 inhibitor is a HER2 antibody or antibody fragment, or a smallmolecule, which binds to and inhibits the HER2 receptor. In variousembodiments, the HER2 antibody may inhibits HER2 ectodomain cleavage,may block ligand activation of a HER receptor, or may inhibit HER2dimerization.

Known HER2 inhibitors are tyrosine kinase inhibitors (Lapatinib) andmonoclonal antibodies (Trastuzumab and Pertuzumab). HER2 receptor is atransmembrane receptor; it has an extracellular binding component, atransmembrane component and an intracellular tyrosine kinase component.Tyrosine kinase inhibitors bind to the tyrosine kinase domain in theHER2 and stops activation of the signaling pathway. Monoclonalantibodies bind to the extracellular component of the HER2 and stop thereceptor activation.

A convenient approach is to obtain a concentration dependent responsefor an inhibitor by performing a dose dependent curve study. By way ofexample, Lapatinib can be used from about 0.01 nM to about 10 nM, in oneembodiment from about 0.05 nM to about 5 nM, in another embodiment fromabout 0.1 nM to about 1 nM. The other inhibitors can be used at the sameconcentrations as Lapatinib or modified as appropriate. The sensitivityof the HER2 variant toward a particular inhibitor can be convenientlymeasured by an increasing light emission as compared to a negativecontrol (i.e., an assay cell exposed to vehicle alone without theinhibitor).

In certain embodiments, the cells express a knock down or knockout ofendogenous HER 1, HER2 and/or HER3. In a particular embodiment the assaycells have a DKO of HER1 and HER2. In certain embodiments, the knockdown is a genomic modification of at least a portion of the desired HERgene. In certain embodiments, the genomic modification is performedusing CRISPR-CAS9 technology. In certain embodiments, the genomicmodification is performed using TALENs or recombination technology.

In one aspect, the present invention provides an assay to test patientvariants of the HER2 gene, preferably as identified by next generationsequencing (NGS), thus determining potentially hyperactive and/orinhibitor resistant mutations. When cells are treated with a HER2inhibitor and HER2 is inhibited, activation of the JNK signaling pathwayis prevented which results in decreasing the growth and replication ofthe cell, thereby inhibiting cancer cells.

In certain embodiments, the present assay may be used to determinewhether a patient HER2 variant will respond to a specific inhibitor. Thespecific variant is determined from a patient’s biological sample. Themethod involves preparing a cDNA containing a HER2 variant from apatient followed by transfecting the cDNA into a cell. Depending uponthe particular cell line and other conditions, if any endogenous HER2can be produced by the assay cells, the cells can undergo a genomicmodification for gene deletion (knockout) or knockdown to reduce orprevent interference with the assays of the invention. In anotherembodiment, if the assay cells do not express the endogenous HER2 gene,then no such genomic modification will be needed. It also may bedesirable to knockout or knockdown other HER receptors to improve assayinterference, e.g., HER1. After determining the activity of the HER2variant of a patient, the physician can use this information to adjusttherapy. Appropriate therapy may include surgery, radiation,chemotherapy, immunotherapy, hormone therapy, targeted therapy, and thelike. For example, if the HER2 variant is constitutively active and itsactivity is independent of ligand, then HER2 inhibitors should not beused. If the HER2 variant activity depends on ligand, then HER2inhibitors should be used.

In some embodiments, the biological sample is from patients selectedfrom: blood, serum, and tumor tissue. The biological sample can betissue or cells from a breast tumor. In some embodiments, the biologicalsample may be freshly isolated. In some embodiments, the biologicalsample may be frozen. In some embodiments, the biological sample may befixed. The biological sample can be processed to obtain HER2 genomicinformation and the HER2 variants can be sequenced using knowtechniques, e.g., NGS. The genomic information can then be used togenerate polynucleotides that can then be used to transfect the assaycells. In an alternate embodiment, the HER2 polynucleotides can beisolated directly from the biological sample and used to transfect theassay cells.

Sample preparation includes isolation of nucleic acids (e.g., DNA,mRNA). These isolation procedures involve separation of nucleic acidsfrom insoluble components (e.g., cytoskeleton) and cellular membranes.In one embodiment, biological tissues or cells are treated with a lysisbuffer solution prior to isolation of nucleic acids. A lysis buffersolution is designed to lyse tissues, cells, lipids and otherbiomolecules potentially present in the raw tissue samples. Generally, alysis buffer of the present invention may contain a chemical agent thatincludes one or more of the following ingredients: (i) chaotropic agents(e.g., urea, guanidine thiocyanide, or formamide); (ii) anionicdetergents (e.g., SDS, N-lauryl sarcosine, sodium deoxycholate, olefinesulphates and sulphonates, alkyl isethionates, or sucrose esters); (iii)cationic detergents (e.g., cetyl trimethylammonium chloride); (iv)non-ionic detergents (e.g., Tween®-20, polyethylene glycol sorbitanmonolaurate, nonidet P-40, Triton® X-100, NP-40, N-octyl-glucoside); (v)amphoteric detergents (e.g., CHAPS,3-dodecyl-dimethylammonio-propane-1-sulfonate, lauryldimethylamineoxide); or (vi) alkali hydroxides (e.g., sodium hydroxide or potassiumhydroxide). Suitable liquids that can solubilize the cellular componentsof biological samples are regarded as a lysis buffer for purposes ofthis application.

In another embodiment, a lysis buffer may contain additional substancesto enhance the properties of the solvent in a lysis buffer (e.g.,prevent degradation of nucleic acid components within the raw biologicalsamples). Such components may include RNAse inhibitors, DNAseinhibitors, and the like. RNAse inhibitors include common commerciallyavailable inhibitors such as SUPERase.InTM (Ambion, Inc. Austin, Tx),RNAse Zap® (Ambion, Inc. Austin, Tx), Qiagen RNase inhibitor (Valencia,CA), and the like.

Nucleic acids, such as mRNA or DNA, can be conveniently extracted frombiological samples using standard extraction methods that are known inthe art. Standard extraction methods include the use of a chemical agentsuch as guanidinium thiocyanate, phenolchloroform extraction,guanidine-based extraction, and the like. Commercial nucleic acidextraction kits may be employed. For example, RNeasy Fibrous Tissue MiniKit from Qiagen (Valencia, CA) and RNAimage Kit from GenHunterCorporation (USA).

The technique of “polymerase chain reaction” or “PCR” as used hereingenerally refers to a procedure wherein minute amounts of a specificpiece of nucleic acid, RNA and/or DNA, are amplified as described inU.S. Pat. No. 4,683,195. Generally, sequence information from the endsof the region of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers may coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (StocktonPress, NY, 1989). As used herein, PCR is considered to be one, but notthe only, example of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample, comprising the use of a knownnucleic acid (DNA or RNA) as a primer and utilizes a nucleic acidpolymerase to amplify or generate a specific piece of nucleic acid or toamplify or generate a specific piece of nucleic acid which iscomplementary to a particular nucleic acid.

Any suitable sequencing method can be used according to the invention,Next Generation Sequencing (NGS) technologies being preferred. ThirdGeneration Sequencing methods might substitute for the NGS technology inthe future to speed up the sequencing step of the assay. Forclarification purposes: the terms “Next Generation Sequencing” or “NGS”in the context of the present invention mean all novel high throughputsequencing technologies which, in contrast to the “conventional”sequencing methodology known as Sanger chemistry, read nucleic acidtemplates randomly in parallel along the entire genome by breaking theentire genome into small pieces. Such NGS technologies (also known asmassively parallel sequencing technologies) are able to deliver nucleicacid sequence information of a whole genome, exome, transcriptome (alltranscribed sequences of a genome) or methylome (all methylatedsequences of a genome) in very short time periods, e.g. within 1-2weeks, preferably within 1-7 days or most preferably within less than 24hours and allow, in principle, single cell sequencing approaches.Multiple NGS platforms which are commercially available or which arementioned in the literature can be used in the context of the presentinvention e.g. those described in detail in Zhang et al. 2011: Theimpact of next-generation sequencing on genomics. J. Genet Genomics 38(3), 95-109; or in Voelkerding et al. 2009: Next generation sequencing:From basic research to diagnostics. Clinical chemistry 55, 641-658.

The assay cells of the invention are capable of expressing a reporterregulated by HER2. In some embodiments, the test cell is a eukaryoticcell. In some embodiments the assay cells are mammalian cells, such asrat, mouse, hamster, monkey and human cells. In some embodiments, thetest cell may be a primary cell or a cell line. In another embodiment,an assay cell is a non-cancerous cell. In another embodiment, an assaycell is derived from a cell line. In another embodiment, an assay cellis amenable by transfection. In another embodiment, an assay cell isamenable by transient transfection. In another embodiment, an assay cellis a cell in which the expression of one or more endogenous genes havebeen reduced or eliminated by any molecular method. For example, in someembodiments it may be desirable to knockdown or knockout endogenous HER1and/or HER2. Specific examples of cells useful in the assays of theinvention (i.e., before modifying the cells with the constructsdescribed herein, also known as “parent cells”) include HEK293 (humanembryo kidney), MCF-7 (human breast cancer), Hela (human cervixepithelial carcinoma), HT29 (human colon adenocarcinoma grade II), A431(human squamous carcinoma), IMR 32 (human neuroblastoma), K562 (humanchronic myelogenous leukemia), U937 (human histiocytic lymphoma),MDA-MB-231 (Human breast adenocarcinoma), SK-N-BE(2) (humanneuroblastoma), SH-SY5Y (human neuroblastoma), HL60 (human promyelocyticleukemia), CHO (hamster Chinese ovary), COS-7 (monkey African greenkidney, SV40 transformed), S49 (mouse lymphoma), Ltk (mouseC34/connective tissue), NG108-15 (mouse neuroblastoma x Rat gliomahybrid), B35 (rat nervous tissue neuronal), B50 (rat nervous tissueneuronal), B104 (rat nervous tissue neuronal), C6 (rat glial tumor),Jurkat (human leukemic T cell lymphoblast), BHK (hamster Syrian kidney),Neuro-2a (mouse albino neuroblastoma), NIH/3T3 (mouse embryofibroblast), A549 (human adenocarcinoma alveolar epithelial), Be2C(human neuroblastoma), SW480 (human lymph node metastasis), Caco2 (humanepithelial colorectal adenocarcinoma), THP1 (human acute monocyteleukemia), IMR90 (human lung fibroblast), HT1080 (human fibrosarcoma),LnCap (human prostate adenocarcinoma), HepG2 (human liver carcinoma)PC12 (rat pheochromocytoma), or SKBR3 (human breast cancer) cells. Inanother embodiment, an assay cell is U20S cell. In another embodiment,an assay cell is NCI60 cell lines, such as, A549, EKVX, T47D, HT29.

HEK 293 cells are a specific cell line originally derived from humanembryonic kidney cells grown in tissue culture and are a preferredparent cell line. HEK 293 cells have been widely used in cell biologyresearch for many years, because of their reliable growth and propensityfor transfection.

In the assays using HER2 inhibitors, the inhibitors that are exposed tothe assay cells are typically solubilized or suspended in a vehicle. Acontrol is typically performed with vehicle without the inhibitor.Depending on the compound to be utilized as an inhibitor in the assay,suitable vehicles include dimethylsulfoxide (“DMSO”), dimethylformamide(“DMF”), water, aliphatic alcohols, and mixtures thereof.

According to some embodiments, there is provided a kit for determiningHER2 activity in a patient or for determining a patient’s response toHER2 inhibitors. In some embodiments, there is provided a kit forassessing patient specific mutations.

In some embodiments, the invention provides a kit for determining themolecular cancer profile in a subject, by identifying patient specificHER2 variants. In another embodiment, the kit comprises at least onemeans of detecting a reporter gene. In some embodiments, the kitcontains one or more of: a substrate or container for holding nucleicacid molecules and/or test cells, directions for carrying out theassay(s), test cells, transfection reagents, or any combination thereof.

Compositions of the present invention may, if desired, be presented inan article of manufacture, which may contain diagnostic reagents andprinted instructions for use. The kit may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale of assayproducts.

The assays are performed in a vessel capable of holding the cells andreagents and not interfering with assay results. In some embodiments theassay cells adhere to the surfaces of the vessel, e.g., the surfaces ofassay plate wells. In some embodiments the assays are miniaturized anduse multi-well plates known in the art. In certain embodiments, thepresent assay can be conveniently used in a 96 well plate, but can alsobe adopted for high throughput in 384 well plates or 1536 well plates.One skilled in the art will be able to easily optimize the well platesto suit throughput necessity. Plates for the assays are typically madeof a polymer, e.g., polystyrene and the like. In some embodiments theplates are surface treated to facilitate adherence of the assay cells tothe wells of the plate, such treatment is commonly referred to as“tissue culture treated”. The surface treatment is typically an oxygenplasma discharge that renders the surface of the wells more hydrophilic.In some embodiments dispensing the cells and/or reagents for the assaysinto the wells of the plates is automated. In some embodiments the cellsand/or reagents are dispensed continuously at a high speed. In oneembodiment an acoustic liquid dispenser is used to dispense thereagents, e.g., HER2 inhibitors.

Any discussion of the content of references cited herein is intendedmerely to provide a general summary of assertions made by the authors ofthe references, and does not constitute an admission as to the accuracyof the content of such references. The following examples are providedto further illustrate various preferred embodiments and techniques ofthe invention. It should be understood, however, that these examples donot limit the scope of the invention described in the claims. Manyvariations and modifications are intended to be encompassed within thespirit and scope of the invention.

EXAMPLES Example 1 Next-Generation Sequencing to Interrogate Mutation ofTumor

Genetic change or mutation of genes is one of the mechanisms to acquireresistance in breast cancer during targeted therapy. To survey themutation status of HER2 genes, whole-exome sequencing of the tumor wasperformed. Cancer tissue was isolated from formalin fixed paraffinembedded (“FFPE”) blocks of tumor. Genomic DNA was extracted and wasused to prepare a library for next-generation sequencing. The identifiedmutations, which caused changes in the amino acid sequence, wereselected to examine their functional effect on the genes in thecell-based assay of the invention. The patient gene carrying theidentified mutation is constructed using PCR mediated overlappingextension in a format of linear expression cassette.

Example 2 Construction of Linear Expression Cassette of Human HER2Receptor

In order to study the effect of unknown mutations in human HER2 gene,linear expression cassettes were generated, which contained CMV promotercontrolling expression of HER2 coding sequence followed by terminatorand polyadenylation signal. To do so, overlapping extension PCR wasemployed to construct the linear expression cassette using an expressionplasmid of human HER2 as PCR template. Using this method, theconstruction of a linear expression cassette takes around 4-8 hours.However, the traditional cloning method to generate an expressionplasmid takes around 2-4 days. Therefore, making patient gene(s) in alinear expression cassette format is highly advantageous for a clinicaldiagnostic test because of its quick turn-around time.

Construction of Expression Plasmids of Human HER1, HER2 and HER3

cDNA plasmids containing human HER1, HER2 and HER3 genes were purchased(Dharmacon). The coding sequence of these genes was amplified by thePCR. NheI and XhoI restriction enzyme sites were inserted into forwardand reverse primers respectively for the purpose of cloning of HER1 andHER2. NheI and XbaI restriction enzyme sites were inserted into forwardand reverse primers respectively for the purpose of cloning of HER3. ThePCR products containing the coding sequences of these human genes weresub-cloned into the pcDNA3.1 (+) using NheI and XbaI or XhoI restrictionenzymes. The nucleotide sequences of human HER1, HER2 and HER3 wereverified by DNA sequencing. The human HER2 expression plasmid was usedas PCR template to construct the linear expression cassettes ofwild-type HER2 or mutated forms of HER2. The expression plasmids of HER1and HER3 were used in the identification and construction of a reportersystem for HER2 assay.

Generation of Linear Expression Cassette of Human Normal and MutatedHER2

A linear expression cassette of human wild-type HER2 was generated byPCR using UF-CMV forward and BGH-UR reverse primers. The amplifiedproducts were gel-purified. The DNA concentration was quantitated by theoptical density at 260 nm using a Nanodrop spectrophotometer.

A linear expression cassette of mutated HER2 was generated by a PCRmediated overlapping extension method. A pair of forward and reverseprimers containing the targeted mutations was designed. The mutatedcodon (3 nucleotides) was located in the middle of a primer flanked by18 nucleotides in each side. Two separate PCR procedures, named as PCR#1and PCR#2 in FIG. 1 , were performed using UF-CMV forward and mutatedreverse primers, or mutated forward and BGH-UR primers. The PCR productswere purified by ExoSAP-IT® (Affymetrix) to remove unconsumed dNTPs andprimers. These two treated PCR products were mixed together followed bydilution with water. A second round of PCR was performed using thediluted PCR mixture as template, and UF and UR primers (FIG. 1 ). Theamplified products were gel-purified. The DNA concentration wasquantitated by the optical density at 260 nm using Nanodrop. Thetargeted mutations were incorporated in the HER2 gene using this PCRmediated overlapping extension method.

Example 3 Identification of Transfection Method Comparison of Forward orReverse Transfection Protocols

In order to test the possibility of using “reverse transfection” methodto save time (typically about a day), a reporter plasmid, CDH1-luc,containing active CDH1 promoter was used. Since our clinical assayemployed the linear expression cassette to express the patient ESR1 genewhich contains a mutation found in the tumor, PCR was performed togenerate a linear version of CDH1-luc. This linear reporter wasgel-purified. The DNA concentration was quantitated by the opticaldensity at 260 nm using Nanodrop.

To compare the “forward transfection” and “reverse transfection”protocols, HEK 293 cells were transiently transfected with 100 ng ofpurified linear reporter using TransIT-293 transfection reagent (MirusBio). This transfection reagent was optimized to give maximumtransfection performance in HEK 293 cells. The transfection efficiencywas measured by Nano-Glo® Luciferase assay (Promega) 24 hourspost-transfection (FIG. 2 ). Significant luciferase activity wasdetected over the background signal using either forward or reversetransfection protocol. There is no substantial difference of luciferasesignal between these two protocols. Therefore, these resultsdemonstrated that a “reverse transfection” protocol can be used totransiently transfect linear DNA into HEK 293 cells.

Example 4 Generation of Double Knockout Cells - HER1 and HER2 Receptors

The expression of HER1, HER2, and HER3 was characterized in HEK293cells.

Expression of HER1, HER2 and HER3 in the HEK293 Cells

In order to examine the expression of HER1, HER2 and HER3 receptors,HEK293 cells were first transfected with pcDNA (as a negative control),or expression constructs of HER1, HER2 or HER3. Cellular lysates wereprepared 48 hours after transfection. Immunoblots were performed usinganti-Her1, anti-Her2 and anti-Her3 antibodies (FIG. 3 ). When the cellswere transfected with the expression plasmids of these receptors, theirexpression were detected in all cases. When the cells were transfectedwith the control plasmid (pcDNA), endogenous HER1 and HER2 receptorswere detected in the HEK293 cells but no HER3 receptor was detected.This result indicates that a preferred embodiment is to deleteendogenous HER1 and HER2 receptors for the assay of the invention.

Knockout of HER2 by CRISPR Technology

In order to knockout the HER2 receptor in the genome of HEK293 cells,CRISPR knockout constructs designed specifically for human HER2 wereobtained from Santa Cruz Biotechnology. The constructs containing gRNAsequences direct the Cas9 protein to induce a site-specific doublestrand break in the HER2 genomic region, which results in the deletionafter repairing. To examine the deletion, primers flanking the gRNAtargeting region were designed. These primers were used to performgenomic PCR to reveal the genomic status.

In transient transfection, HEK293 cells were transfected with the CRISPRconstructs. Genomic DNA was prepared 48 hours after transfection. PCRwas performed using the primers flanking the targeted area. In FIG. 4A,a smaller PCR band was detected in the cells transfected with CRISPRconstructs. This result shows that these HER2 specific CRISPR constructsare capable of inducing double strand DNA break which results indeletion.

In order to generate stable knockout HER2 cells, the CRISPR constructswere transiently transfected into the HEK293 cells. The transfectedcells were singly plated in 96-well plate 24 hours post-transfection.Seven single cells were chosen and expanded. Genomic DNA was prepared.PCR was performed using the primers flanking the targeted area. In FIG.4B, clone #1 showed only the smaller PCR band, which indicated that itis potentially a homozygous HER2 knockout clone. To confirm the HER2knockout status of clone #1, cellular extract was prepared. Immunoblotwas performed using anti-Her2 antibody (FIG. 5 ). Expression of HER2receptor was not detected in the clone #1. This result confirmed thatclone #1 is a complete knockout of HER2 receptor.

As shown in FIG. 3 , HEK293 cells expressed both HER1 and HER2receptors. The same procedure was repeated to knockout HER1 receptor inthis HER2 KO clone #1. To do so, CRISPR knockout constructs designedspecifically for human HER1 were obtained from Santa Cruz Biotechnology.The constructs containing gRNA sequences direct the Cas9 protein toinduce a site-specific double strand break in the HER1 genomic region,which results in the deletion after DNA repairing. The CRISPR constructswere transiently transfected into the clone #1 cells. The transfectedcells were singly plated in 96-well plate 24 hours post-transfection.100 single cell clones were screened by genomic PCR using the primersflanking the targeted area. Only 4 single cell clones, #10, #19, #27 and#53, showed homozygous knockout of HER1 in the genome. The deletion ofHER1 receptor was confirmed by the immunoblot assay. In FIG. 6 , no HER1receptor was detected in clone #10, #19 and #23. However, clone #53showed a truncated form of HER1 receptor. Cellular lysates from theseclones were also used to check the expression of HER2 receptor. All ofthem showed no expression of HER2 receptor. Clone #10, #19 and #27represented homozygous knockout of both HER1 and HER2. Double knockoutclone #27 (HEK293 DKO#27) was used in the following examples to generatethe reporter cell to measure the activity of HER2 receptor.

Example 5 Identification of Reporters to Measure Activity of HER2 byOverexpression of HER2 Receptor Overexpression of HER2 Receptor

In order to identify a reporter to measure the activity of HER2receptor, the HEK293 cells were transfected with different reporterconstructs, including ERK-Luc, FRE-Luc, JNK-Luc and NFkB-Luc, in theabsence or presence of HER2 expression plasmid. After 24 hours, theluciferase reporter activity of the transfected cells was measured byNano-Glo® Luciferase Assay (Promega). In all the reporters tested,increased reporter activity was found in the presence of HER2 receptor(FIG. 7 ). NFkB-Luc reporter showed the best signal to background ratioamong the reporters tested.

Overexpression of HER2 Receptor Treated With Lapatinib

In order to characterize the specificity of activation of thesereporters upon HER2 expression, the same transfection experiment wasperformed as described in Example 5a. After 24 hours, the transfectedcells were treated with 10 nM of lapatinib, a tyrosine kinase inhibitorof HER2, for 4 hours. The luciferase reporter activity of thetransfected cells was measured by Nano-Glo® Luciferase Assay (Promega).In all the reporters tested, increased reporter activity was found inthe presence of HER2 receptor (FIG. 8 ). However, lapatinib did notinhibit these increased reporter activities. This observation shows thatthe increased reporter activities upon HER2 expression are independentof tyrosine kinase activity of HER2 receptor.

Overexpression of Wild-Type, Constitutively Active and Kinase-Dead Formsof HER2 Receptor

In order to characterize the specificity of activation NFkB-Luc reporterupon HER2 expression, a similar experiment was performed by transfectingthe HER2 expression plasmids encoding wild-type, constitutively activeor kinase-dead forms of HER2. After 24 hours, the luciferase reporteractivity of the transfected cells was measured by Nano-Glo® LuciferaseAssay (Promega). In all forms of HER2 tested, increased reporteractivity was found (FIG. 9 ). The reporter activity was similar when thecells were transfected with either wild-type or kinase-dead form ofHER2. This observation shows that the increased reporter activities uponwild-type HER2 expression are independent of tyrosine kinase activity ofHER2 receptor.

Overexpression of Wild-Type HER2 Receptor With Lapatinib, Trastuzumaband Pertuzumab

In order to further characterize the specificity of activation ofNFkB-Luc reporter upon HER2 expression, the HEK293 cells weretransfected with the wild-type HER2 expression plasmid followed bytreatment of HER2 inhibitors for 4 hours. The luciferase reporteractivity of the transfected cells was measured by Nano-Glo® LuciferaseAssay (Promega). Overexpression of HER2 receptor activated the NFkB-Lucreporter but none of the HER2 inhibitors block this activation (FIG. 10). This observation indicated that HER2 expression per se is not adesirableoption to measure its receptor activity and its response towardthe inhibitors.

Example 6 Identification of Reporters to Measure Activity of HER2 byOverexpression of HER1, HER2 and HER3 Receptors Overexpression of HER1,HER2 and HER3 Receptors

In order to identify reporters to measure the activity of HER2 receptor,expression plasmids of HER1, HER2 and HER3 were co-transfected withdifferent reporter constructs, including ERK-Luc, JNK-Luc and NFkB-Luc,into the HEK293 cells. After 24 hours, the transfected cells weretreated with either EGF (HER1 receptor ligand) or NRG1 (HER3 receptorligand) for 6 hours. The luciferase reporter activity of the transfectedcells was measured by Nano-Glo® Luciferase Assay (Promega).Interestingly, ERK-Luc reporter was activated upon the treatment of HER3ligand (FIG. 11A). JNK-Luc reporter was activated when the transfectedcells were treated with either HER1 or HER3 ligand (FIG. 11B). However,NFkB-Luc reporter showed no activation upon addition of either HER1 orHER3 ligand (FIG. 11C). This observation shows that either ERK-Luc orJNK-Luc can be used to measure activity of HER2 receptor.

Overexpression of HER1 and Wild-Type or Kinase-Dead HER2 Receptors

In order to characterize the specificity of activation of JNK-Lucreporter upon treatment of HER1 ligand, JNK-Luc was co-transfected withexpression plasmids of HER1 and wild-type or kinase-dead HER2 receptorsinto the HEK293 cells. After 24 hours, the transfected cells weretreated with EGF (HER1 receptor ligand). The luciferase reporteractivity of the transfected cells was measured by Nano-Glo® LuciferaseAssay (Promega). In FIG. 12 , no difference was found between the cellstransfected with wild-type HER2 and kinase-dead HER2. In both case, theEGF treatment effectively activated the JNK-Luc reporter. Thisobservation indicated that such activation was independent of HER2′skinase activity.

Overexpression of HER3 and Wild-Type or Kinase-Dead HER2 Receptors

In order to characterize the specificity of activation of ERK-Luc andJNK-Luc reporters upon treatment of HER3 ligand, ERK-Luc or JNK-Luc wasco-transfected with expression plasmids of HER3 and wild-type orkinase-dead HER2 receptors into the HEK293 cells. After 24 hours, thetransfected cells were treated with NRG1 (HER3 receptor ligand). Theluciferase reporter activity of the transfected cells was measured byNano-Glo® Luciferase Assay (Promega). In FIG. 13A, no difference inERK-Luc reporter activity was found between the cells transfected withwild-type HER2 and kinase-dead HER2 after the treatment of NRG1. In bothcase, the NRG1 treatment effectively activated the ERK-Luc reporter.This observation indicated that such ERK-Luc activation was independentof HER2′s kinase activity. However, the activity of JNK-Luc reporter wasreduced in the cells transfected with kinase-dead HER2 compared to thecells transfected with wild-type HER2 (FIG. 13B). This observationindicated that overexpression of HER2 and HER3 receptors responded toNRG1 treatment, which activated JNK-Luc reporter in a HER2′s kinasedependent manner.

Overexpression of HER3 and HER2 Receptors - NRG1 Dose

In order to examine the dose response of NRG1 in the activation ofJNK-Luc reporters, JNK-Luc with expression plasmids of HER3 andwild-type HER2 receptors were co-transfected into the HEK293 cells.After 24 hours, the transfected cells were treated with a serialdilution of NRG1 (HER3 receptor ligand) for 6 hours. The luciferasereporter activity of the transfected cells was measured by Nano-Glo®Luciferase Assay (Promega). In FIG. 14 , a dose dependent activation ofJNK-Luc was shown when the cells expressed HER2 and HER3 receptors.

Modification of JNK-Luc Reporter

In the previous example, the JNK-Luc reporter contained 3 copies of AP-1binding site. We observed around 2.6 fold induction of reporter activitywhen the transfected cells were treated with 100 ng/ml of NRG1 (FIG. 14). In order to enhance the activation of reporter activity, the AP-1binding site was increased from 3 copies to 6 copies. 6X JNK-Luc wasco-transfected with expression plasmids of HER3 and wild-type HER2receptors into the HEK293 cells. After 24 hours, the transfected cellswere treated with 100 ng/ml of NRG1 (HER3 receptor ligand) for 6 hours.The luciferase reporter activity of the transfected cells was measuredby Nano-Glo® Luciferase Assay (Promega). In FIG. 15 , the 6 X JNK-Lucreporter was activated by the treatment of NRG1 only when both HER2 andHER3 receptors were expressed (FIG. 15 ). Approximately a 4.2 foldinduction of reporter activity was found.

Example 7 Validation of Reporters to Measure Activity of HER2 byOverexpression of HER2 and HER3 Receptors in DKO#27 Cells

In example 6, 6X JNK-Luc reporter was used to measure activity of HER2in the HEK293 cells transfected with the expression constructs of HER2and HER3. Since these cells expressed HER2 receptor, the same reporterexperiment was performed using DKO#27 cells, in which both HER1 and HER2reporters were deleted. This reporter system was also validated usinginhibitors of HER2 and mutants of HER2 receptors.

6X JNK-Luc Reporter in the DKO#27 Cells - NRG1 Dose

In example 4, the generation double knockout cells (HEK293 DKO#27) ofHER1 and HER2 receptors was described. No protein expression of HER1 andHER2 receptors was detected by immunoblot assays. In order tocharacterize whether these cells are desirable for use in the assay ofthe invnetion, 6X JNK-Luc was co-transfected with expression plasmids ofHER3 and wild-type HER2 receptors. After 24 hours, the transfected cellswere treated with a serial dilution of NRG1 (HER3 receptor ligand) for 6hours. The luciferase reporter activity of the transfected cells wasmeasured by Nano-Glo® Luciferase Assay (Promega). In FIG. 16 , a dosedependent activation of JNK-Luc was observed in these transfected cells.

6X JNK-Luc Reporter in the DKO#27 Cells - HER2 Inhibitors

HER2 inhibitors include tyrosine kinase inhibitors (Lapatinib) andmonoclonal antibodies (Trastuzumab and Pertuzumab). HER2 receptor is atransmembrane receptor; it has an extracellular binding component, atransmembrane component and an intracellular tyrosine kinase component.Tyrosine kinase inhibitors bind to the tyrosine kinase domain in theHER2 and stops activation of the signaling pathway. Monoclonalantibodies bind to the extracellular component of the HER2 and stop thereceptor activation. [this paragraph is already in the detaileddescription]

In order to validate the requirement of HER2 receptor in the activationof 6X JNK-Luc reporter in response to NRG1 treatment, the DKO#27 cellswere transfected with the wild-type HER2 and HER3 expression plasmidsfollowed by treatment of HER2 inhibitors for 6 hours. The luciferasereporter activity of the treated cells was measured by Nano-Glo®Luciferase Assay (Promega). In the absence of inhibitors, NRG1 activatedthe reporter (FIG. 17 ). Such activation was abolished when these cellswere treated with the inhibitors of HER2. This observation shows thatthe activation was going through HER2 receptor. The 6X JNK-Luc reporteris capable of measuring the activity of HER2 and its response to theinhibitors.

6X JNK-Luc Reporter in the DKO#27 Cells - HER2 Mutants, IncludingWild-Type, Kinase-Dead and Constitutively Active Forms

In order to validate that this 6X JNK-Luc reporters has the capacity ofmeasuring HER2 receptor activity, the reporters were co-transfected withexpression plasmids of HER3 and wild-type, constitutively active orkinase-dead HER2 receptors into the DKO#27 cells. After 24 hours, thetransfected cells were treated with a serial dilution of NRG1. Theluciferase reporter activity of the transfected cells was measured byNano-Glo® Luciferase Assay (Promega). The transfected cells expressedwild-type form of HER2 receptor showed a dose dependent response uponthe treatment of NRG1 (FIG. 18 ). When the cells were transfected withthe constitutively active form of HER2 receptor, the reporter activitywas activated without the treatment of NRG1. However, no activation ofreporter was detected when the cells were transfected with kinase-deadform of HER2 receptor. These observations show that 6X JNK-Luc reportercan be used to measure the activity of HER2 receptor.

Example 8 Construction and Characterization of Stable Reporter CellsContaining 6X JNK-Luc Reporter Plasmid

The 6X JNK-Luc reporter system has been validated by multipleapproaches. This reporter system can be used to measure the HER2receptor activity, its response toward the inhibitors and the mutationeffect on the activity of HER2 receptor. In a preferred embodiment thisreporter system is used as a clinical diagnostic test. To improve thereporter system for use in a clinical setting, reporter cells weregenerated inwhich the 6X JNK-Luc plasmid was stably integrated in thegenome of DKO#27 cells.

Selection of Stable Cell Clones Carrying 6X JNK-Luc Plasmid

Since the 6X JNK-Luc plasmid did not contain a selectable marker, the 6XJNK-Luc was co-transfected with pIRES-hygB, which contained hygomycin Bresistance gene, in 5:1 ratio into DKO#27 cells. Antibiotic, hygromycinB, was added to the culture medium 48 hours post-transfection. Any cellssurviving through the selection process suggested that those cells notonly expressed hygomycin B resistance gene, which was encoded inpIRES-hygB plasmid, but also carried the 6X JNK-Luc reporter plasmid asthe co-transfection of 6X JNK-Luc with pIRES-hygB was performed in 5:1ratio.

Characterization of Single Cell Clones After Hygomycin B Selection

Forty single cell clones were selected from the selection plate and wereexpanded. In order to confirm the integration of the 6X JNK-Luc reporterplasmid, these single cell clones were transfected with the expressionplasmids of HER2 and HER3 receptors. After 24 hours, the transfectedcells were stimulated with NRG1 for 6 hours. The luciferase reporteractivity was measured by Nano-Glo® Luciferase assay (Promega) (FIG.19A). Four out of forty clones showed detectable basal luciferasereporter activity in the absence of NRG1. Upon treatment with the NRG1,seven out of forty clones showed activation of the luciferase reporteractivity (FIG. 19B). Among these 7 clones, clone #4, #11, #28 and #33showed the best fold of induction (>2 folds). Therefore, these fourclones were used for further analysis.

Comparison of Reporter Activity in the Clone #4, #11, #28 and #33 Uponthe Treatment of NRG1

The 6X JNK-Luc single cell clones #4, #11, #28 and #33were transfectedwith the expression plasmids of HER2 and HER3 receptors. After 24 hours,the transfected cells were stimulated with the NRG1 for 6 hours. Theluciferase reporter activity was measured by Nano-Glo® Luciferase assay(Promega) (FIG. 20A). No reporter activity was detected in the clone #4.Clone #11 has the highest basal reporter activity among those tested.Upon the treatment of NRG1, clone #28 showed the best fold of activationof reporter among these 4 clones (FIG. 20B). Therefore, clone #28 waschosen for further characterization and validation.

Dose Response Curve of NRG1 Using Clone #28 - HER3 Plasmid and HER2Linear Expression Cassette

In the assay of the invention, expression of patient HER2 receptor is inthe linear expression cassette format instead of plasmid DNA. Therefore,the clone #28 cells were transfected with HER3 expression plasmid andHER2 linear expression DNA. After 24 hours, the transfected cells werestimulated with a serial dilution of NRG1 for 6 hours. The luciferasereporter activity was measured. This clone responded to the treatment ofNRG1 in a dose dependent manner (FIG. 21 ). Therefore, this clone can beused to measure HER2 receptor activity by transfecting linear expressionDNA of HER2.

Dose Response Curve of NRG1 Using Clone #28 - HER3 Plasmid and HER2Linear Expression Cassette of Wild-Type, Constitutively Active orKinase-Dead

In order to validate that the clone #28 cells was capable to measure theHER2 receptor activity, expression plasmids of HER3 were co-transfectedwith wild-type, constitutively active or kinase-dead HER2 receptors.After 24 hours, the transfected cells were treated with a serialdilution of NRG1. The luciferase reporter activity of the transfectedcells was measured by Nano-Glo® Luciferase Assay (Promega). Thetransfected cells expressingwild-type form of HER2 receptor showed adose dependent response upon the treatment of NRG1 (FIG. 22 ). When thecells transfected with the constitutively active form of HER2 receptor,the reporter activity was activated without the treatment of NRG1.However, no activation of reporter was detected when the cells weretransfected with kinase-dead form of HER2 receptor. These observationsshow that the clone #28 cells can be used to measure the activity ofHER2 receptor.

Inhibition of Clone #28 by Inhibitors of HER2

HER2 inhibitors include tyrosine kinase inhibitors (Lapatinib) andmonoclonal antibodies (Trastuzumab and Pertuzumab). HER2 receptor istransmembrane receptors; it has an extracellular binding component, atransmembrane component and an intracellular tyrosine kinase component.Tyrosine kinase inhibitors bind to the tyrosine kinase domain in theHER2 and stops activation of the signaling pathway. Monoclonalantibodies bind to the extracellular component of the HER2 and stop thereceptor activation. [paragraph probably not needed]

In order to validate that the clone #28 reporter cells were capable ofmeasuring the response of HER2 receptor to its inhibitors, these cellswere first transfected with the HER2 linear expression DNA and HER3expression plasmids followed by treatment of HER2 inhibitors for 6 hoursin the presence of NRG1. The luciferase reporter activity of the treatedcells was measured by Nano-Glo® Luciferase Assay (Promega). In theabsence of inhibitors, NRG1 activated the reporter (FIG. 23 ). Suchactivation was abolished when these cells were treated with theinhibitors of HER2. This observation shows that the activation wasmediated through HER2 receptor. The clone #28 cells are capable ofmeasuring the activity of HER2 and its response to the inhibitors.

Example 9 Construction and Validation of Reporter Cells StablyExpressing HER3 Receptor Selection of Stable Cell Clones Expressing HER3Receptor

In order to simplify the invention assay, reporter cell clone #28 wasmodified. Selection and generation of single cell clone, which stablyexpressed HER3 receptor, were performed. To do so, expression constructof HER3 receptor was transfected into the reporter cell clone #28.Antibiotic, G418, was added to the culture medium 48 hourspost-transfection. Any cells surviving through the selection processsuggested that expression construct of HER3 was integrated into thegenome.

Characterization of Single Cell Clones After G418 Selection

Eight single cell clones were selected from the selection plate and wereexpanded. In order to confirm the expression of HER3 receptor, thesesingle cell clones were transiently transfected with the expressionplasmid of HER2. After 24 hours, the transfected cells were treated withNRG1 for 6 hours. The luciferase reporter activity was measured byNano-Glo® Luciferase assay (Promega) (FIG. 24A). Five out of eightclones showed pronounced luciferase reporter activity upon addition ofNRG1. Among these 5 clones, clone #1 showed the best fold of induction(FIG. 24B). Therefore, this clone was used for further analysis.

The HER2 Reporter Cells (Clone #1) to Measure Activities of WT, CA andKD of HER2 Receptor in the 384-Well Plate

Clone #1 is alternately referred to as the “HER2 reporter cells”. Thesecells were transiently transfected with the wild-type, constitutivelyactive or kinase-dead forms of HER2 receptors in the 384-well format.After 24 hours, the transfected cells were treated with NRG1. Theluciferase reporter activity of the transfected cells was measured byNano-Glo® Luciferase Assay (Promega). The cells with no expression ofHER2 receptor did not show reporter activity and did not respond to NRG1(FIG. 25 ). The reporter activity of the transfected cells expressingthe wild-type form of HER2 receptor was activated upon treatmentwithNRG1 (FIG. 25 ). When the cells were transfected with theconstitutively active form of HER2 receptor, the reporter activity wasactivated in the absence of NRG1. However, no activation of reporter wasdetected when the cells were transfected with the kinase-dead form ofHER2 receptor. These observations show that the HER2 reporter cells canbe used to measure the activity of HER2 receptor.

HER2 Reporter Cells - Transiently Transfected With Linear ExpressionCassette of HER2 Receptor in the 384-Well Plate

The HER2 reporter cells were first transfected with 100 ng of linearexpression cassette of HER2. NRG1 was added to the cells 24 hourspost-transfection. Reporter assay was performed 4, 6 and 24 hours afterthe treatment of NRG1 (FIG. 26 ). All time periods showed adose-dependent response to the treatment of NRG1. In this experiment, abetter response was found when the cells were treated with NRG1 for 4 or6 hours. A robust reporter signal was detected even at 2.5 ng/ml ofNRG1.

Different Stimulation Method

To further reduce the assay turnaround time, different stimulationmethods were explored. The first method evaluated was Example 9d whichwas performed by addition of NRG1 to the cells 24 hourspost-transfection for 4, 6 and 24 hours. Another method was treating thecells with NRG1 when the DNA transfection complex was added to thecells. The reporter activity was measured 24 hours post-transfection(FIG. 27A). This second method is referred to herein as the “all-in-one”method. FIG. 27B shows that the all-in-one method gave a good doseresponse toward the treatment of NRG1. Therefore, the all-in-one methodis a suitable embodiment for the HER2 assay of the invention.

Example 10 HER2 Assay Transfected With Linear Expression Cassette ofWild-Type HER2 in the 384-Well Format

In example 9, the HER2 reporter cells were successfully created and theassay was validated in the 384-well format. In order to test the assayof the invention, the linear expression cassette of wild-type HER2 DNAwas first transfected into the HER2 reporter cells followed by treatmentwith NRG1 and its inhibitors using the “all-in-one” method. Theluciferase reporter activity was measured 24 hours post-transfection.Two different concentrations of inhibitors were experimentallydetermined and used. The low concentration of inhibitor corresponded tothe concentration where treated cells retained more than 50% of HER2activity upon NRG1 stimulation. The high concentration of inhibitorcorresponded to the concentration where treated cells retained less than50% of HER2 activity upon NRG1 stimulation. FIG. 28A shows theluciferase activity of cells transfected with linear HER2 DNA indifferent treatment conditions. The reporter cells responded to NRG1stimulation in a dose dependent manner. FIG. 28B shows the calculatedpercentage of activity of HER2 receptor after treatment with inhibitors.These results clearly show that assay cells of the invention, inparticular the HER2 reporter cells, are capable of measuring theactivity of expressed HER2 receptor and its response toward theinhibitors.

Example 11 Case Studies L755S of HER2

L755S corresponds to a missense mutation of HER2, which changes leucine(L) into serine (S) at amino acid position of 755. In order tofunctionally characterize this mutation, PCR-mediated overlappingextension was employed to construct linear expression cassette of HER2carrying this mutation. The linear expression cassette of mutant HER2DNA was first transfected into the reporter cells followed by treatmentwith NRG1 and its inhibitors using the “all-in-one” method. Theluciferase reporter activity was measured 24 hours post-transfection. InFIG. 29A, increased luciferase activity was observed in the reportercells transfected with this mutant form of HER2 in the absence of NRG1.Therefore, this mutant form of HER2 is partially active and responds totreatment with NRG1.

When these cells were treated with HER2 inhibitors in the presence ofNRG1, reduction of reporter activity was detected for Trastuzumab andPertuzumab. However, the efficacy of these inhibitors was reduced. Inaddition, the L755S of HER2 did not respond to the lapatinib. The resultis summarized in FIG. 29B. Although the mutation partially activatesHER2 receptor, the L755S of HER2 still responds to the inhibitors,including Trastuzumab and Pertuzumab, with reduced efficacy. Clinically,treatment of lapatinib in this patient should be avoided because it maycause clonal expansion of cells, which carry this mutation. Therefore,Trastuzumab and Pertuzumab treatment should be recommended.

P780ins of HER2

P780ins corresponds to an insertion mutation of HER2, which adds 3 aminoacids (GSP) after the amino acid position of 755 (Proline - P). In orderto functionally characterize this mutation, PCR-mediated overlappingextension was employed to construct linear expression cassette of HER2carrying this insertion. The linear expression cassette of mutant HER2DNA was first transfected into the reporter cells followed by treatmentwith NRG1 and its inhibitors using the “all-in-one” method. Theluciferase reporter activity was measured 24 hours post-transfection. InFIG. 30A, pronounced luciferase activity was observed in the reportercells transfected with this mutant form of HER2 in the absence of NRG1.This mutant form of HER2 is constitutively active and its activity isindependent of NRG1.

When these cells were treated with HER2 inhibitors in the presence ofNRG1, very minor reduction of reporter activity was detected forlapatinib. At the high concentration of lapatinib, this mutant HER2receptor still retained more than 75% activity compared to the cellswithout the treatment of inhibitor. The efficacy of this inhibitor wasgreatly reduced. In addition, the P780ins of HER2 did not respond tothese inhibitors, including Trastuzumab and Pertuzumab. The result issummarized in FIG. 30B. Clinically, treatment of Trastuzumab andPertuzumab in this patient should be avoided because it may cause clonalexpansion of cells, which carry this mutation. Since lapatinib showed avery limited activity in the HER2 reporter assay of the invention, ahigher dose of lapatinib should be used. In addition, other targettherapies, such as an mTOR inhibitor or CDK4/6 inhibitor should berecommended in conjunction with the HER2 treatment (Lapatinib) toprevent potential clonal expansion of cells.

T862A of HER2

T862A corresponds to a missense mutation of HER2, which changesThreonine (T) into Alanine (A) at amino acid position of 862. In orderto functionally characterize this mutation, PCR-mediated overlappingextension was employed to construct a linear expression cassette of HER2carrying this mutation. The linear expression cassette of mutant HER2DNA was transfected into the reporter cells followed by treatment withNRG1 and its inhibitors using the “all-in-one” method. The luciferasereporter activity was measured 24 hours post-transfection. In FIG. 31A,increased luciferase activity was observed in the reporter cellstransfected with this mutant form of HER2 in the presence of NRG1.Therefore, this mutant form of HER2 responds to the treatment of NRG1 ina dose-dependent manner.

When these cells were treated with HER2 inhibitors in the presence ofNRG1, reduction of reporter activity was detected for Trastuzumab andPertuzumab. However, the T862A of HER2 did not respond to the lapatinib.The result is summarized in FIG. 31B. Clinically, treatment of lapatinibin this patient should be avoided because it may cause clonal expansionof cells, which carry this mutation. Therefore, Trastuzumab andPertuzumab treatment should be recommended.

Materials and Methods Construction of Expression Plasmids of HER1, HER2and HER3

Human wild-type HER1, HER2 and HER3 cDNA plasmids were ordered from OpenBiosystems (GE Dharmacon). A pair of PCR primers was designed to amplifythe coding region as shown below;

HHER1NHEI F: TGGCTAGCCGCCACCATGCGACCCTCCGGGACGGCC (SEQ ID NO:1)

HHER1 XHOI R: GACTCGAGTCATGCTCCAATAAATTCACT (SEQ ID NO:2)

HHER2 NHEI F: TGGCTAGCCGCCACCATGGAGCTGGCGGCCTTGTGC (SEQ ID NO:3)

HHER2 XHOI R: GACTCGAGTCACACTGGCACGTCCAGACC (SEQ ID NO: 4)

HHER3 NHEI F: TGGCTAGCCGCCACCATGAGGGCGAACGACGCTCTG (SEQ ID NO:5)

HHER3 XBAI R: CCTCTAGATTACGTTCTCTGGGCATTAGC (SEQ ID NO:6)

Restriction enzyme sites of NheI and XhoI were added to the forward andreverse primers respectively for the construction of HER1 and HER2expression plasmids. Restriction enzyme sites of NheI and XbaI wereadded to the forward and reverse primers respectively for theconstruction of HER3 expression plasmid. Coding sequence of wild-typeHER1, HER2 and HER3 was PCR amplified using their corresponding forwardand reverse primers from cDNA plasmid using Q5® high-fidelity DNApolymerase (NEB). The amplified PCR product was run on agarose gels andpurified using DNA gel purification kit from Qiagen. The gel purifiedPCR products and pcDNA3.1 DNA vector were treated with NheI and XhoI orXbaI restriction enzymes at 37° C. for 2 hours. The digested productswere run on agarose gels and purified using DNA gel purification kitfrom Qiagen. The PCR fragments containing coding sequence of wild-typeHER1, HER2 and HER3 were ligated with linearized pcDNA3.1 DNA vectorusing fast ligation kit from NEB. The ligated products were transformedinto Top 10 competent cell (Invitrogen). The transformed competent cellswere selected using LB plate containing ampicillin for 16 hours at 37°C. The ampicillin resistant clones were cultured in 2 mL of LB mediumwith ampicillin for 16 hours at 37° C. DNA was extracted from thebacteria culture using DNA mini-preparation kit from Qiagene. Thewild-type HER1, HER2 and HER3 expression plasmids were confirmed by bothrestriction enzyme digestion and DNA sequencing.

Construction of Linear Expression Cassette of Wild-type HER2 and itsMutants

The following primers were used to construct the linear expressioncassette of wild-type HER2 and its mutants;

UF-CMV F: GCGTTCGCTAAGCGTAGCTAGCGATGTACGGGCCAGATA (SEQ ID NO:7)

UF: GCGTTCGCTAAGCGTAGCTAG (SEQ ID NO:8)

UR-BGH R: TCTGATACGTCTCGACGCACTCTCCCAGCATGCCTGCTATTG (SEQ ID NO:9)

UR: TCTGATACGTCTCGACGCACTC (SEQ ID NO:10)

HER2 L755S F: CCAGTGGCCATCAAAGTGTCCAGGGAAAACACATCCCCC (SEQ ID NO: 11)

HER2 L755S R: GGGGGATGTGTTTTCCCTGGACACTTTGATGGCCACTGG (SEQ ID NO: 12)

HER2 P780INS F: GCTGGTGTGGGCTCCCCAGGTTCTCCCTATGTCTCCCGCCTTCTG(SEQ ID NO:13)

HER2 P780INS R:CAGAAGGCGGGAGACATAGGGAGAACCTGGGGAGCCCACACCAGC (SEQ ID NO:14)

HER2 T862A F: CCCAACCATGTCAAAATTGCAGACTTCGGGCTGGCTCGG (SEQ ID NO:15)

HER2 T862A R: CCGAGCCAGCCCGAAGTCTGCAATTTTGACATGGTTGGG (SEQ ID NO:16)

The linear expression cassette of wild-type HER2 was amplified from thewild-type HER2 expression plasmid using UF-CMV F and UR-BGH R primers.The amplified PCR product was run on agarose gels and purified using DNAgel purification kit from Qiagen.

The linear expression cassettes of mutant forms of HER2 were prepared byPCR mediated overlapping extension. Two rounds of PCR amplification wereperformed. The first round of PCR included two independent PCR usingUF-CMV F and mutation specific R primers or UR-BGH R and mutationspecific F primers to amplify coding region of HER2 into two fragments.The PCR products were then treated with ExoSAP-IT from Affymetrix toeliminate the unincorporated primers and dNTPs. The mixture wasincubated at 37° C. for 15 minutes followed by 80° C. for 15 minutes.Then, 10ul of PCR products from each reaction were added to 30 ul ofwater. 5 ul of the diluted products were used as template to performedsecond round of PCR. In this PCR, UF and UR primers were added to thereaction to construct the linear expression cassette of HER2 carryingthe desired mutation. The amplified PCR product was run on agarose gelsand purified using DNA gel purification kit from Qiagen.

Construction of ERK-Luc, FRE-Luc, JNK-Luc, NFkB-Luc and 6X JNK-LucReporter Constructs

The oligonucleotides corresponding to the binding sites of differenttranscriptional factors to measure the signaling activity of pathwayswere designed and were shown as follow:

3XERK F: TCGAGGGATGTCCATATTAGGAGGATGTCCATATTAGGAGGATGTCCATATTAGGAA (SEQ ID NO: 17)

>3XERK R: AGCTTTCCTAATATGGACATCCTCCTAATATGGACATCCTCCTAATATGGACATCCC(SEQ ID NO:18)

>3XFRE F: TCGAGGATCAAGTAAACAACTATGTAAACAAGATCAAGTAAACAACTATGTAAACAAGATCAAGTAAACAACTATGTAAACAAA (SEQ ID NO: 19)

>3XFRE R: AGCTTTTGTTTACATAGTTGTTTACTTGATCTTGTTTACATAGTTGTTTACTTGATCTTGTTTACATAGTTGTTTACTTGATCC (SEQ ID NO:20)

>3XJNK F: TCGAGTGAGTCAGTGAGTCAGTGAGTCAGA (SEQ ID NO:21)

>3XJNK R: AGCTTCTGACTCACTGACTCACTGACTCAC (SEQ ID NO:22)

>3XNFKB F: TCGAGGGGACTTTCCGGGACTTTCCGGGACTTTCCA (SEQ ID NO: 23)

>3XNFKB R: AGCTTGGAAAGTCCCGGAAAGTCCCGGAAAGTCCCC (SEQ ID NO:24)

The oligonucleotides were re-suspended in TE buffer at 200 µMconcentration. Equal amount of forward and reverse oligonucleotides wereannealed into double strand form by incubated at 95° C. for 10 minutes.After the mixture cooled down to room temperature, 1µl of double strandoligonucleotides was used as insert to ligate with lineralizedpNL3.2reporter construct (Promega). The lineralized vector was prepared bytreating the DNA with XhoI and HindIII restriction enzymes. The ligatedDNA was then transformed into Top10 competent cells (Invitrogen,Carlsbad, CA). These reporter constructs were confirmed by bothrestriction enzyme digestion and DNA sequencing.

In order to construct the 6X JNK-Luc reporter construct,oligonucleotides corresponding to three copies of AP-1 binding site weredesigned to measure the signaling activity of JNK pathway. The sequenceswere shown as follow:

3XJNK F1: CTGAGTCAGTGAGTCAGTGAGTCAGC (SEQ ID NO:25)

3XJNK R1: TCGAGCTGACTCACTGACTCACTGACTCAGGTAC (SEQ ID NO:26)

The oligonucleotides were re-suspended in TE buffer at 200 µMconcentration. Equal amount of forward and reverse oligonucleotides wereannealed into double strand form by incubated at 95° C. for 10 minutes.After the mixture cooled down to room temperature, 1µl of double strandoligonucleotides was used as insert to ligate with lineralizedpNL3.2reporter construct (Promega). The lineralized vector was prepared bytreating the DNA with KpnI and XhoI restriction enzymes. The ligated DNAwas then transformed into Top10 competent cells (Invitrogen, Carlsbad,CA). The 6X JNK-Luc reporter construct was confirmed by both restrictionenzyme digestion and DNA sequencing.

Transfection

For cell transfection experiments, HEK293 cells (ATCC) were plated atdensity of 4-8 x 104 cells per well (96-well plates) or 1-2 x 104 cellsper well (384-well plates) in phenol red-free MEM containing 10% FBS andantibiotics.. Either DNA plasmid or linear DNA was mixed withTransIT-293 transfection reagent (Mirus Bio LLC). Once cells weretrypsinized, DNA transfection mix was added. The cells were thenincubated for 24 or 48 hours.

Immunoblot Assay (Western Blotting)

Cells were collected 48 hours post-transfection, washed in PBS and lysedin ProteoJET mammalian cell lysis reagent (Fermentas) with protease andphosphatase inhibitors (Sigma). Lysates were centrifuged andsupernatants were prepared for SDS-PAGE by addition of sample loadingbuffer (Bio-Rad). Lysates were subjected to 4-12% PAGE (Bio-Rad) andtransferred to Immun-Blot PVDF membrane (Bio-Rad) per manufacturer’srecommendations. Membranes were blocked in 5% milk/TPBT at roomtemperature for 1 hour. Membranes were probed with anti-HER1, anti-HER2,anti-HER3 and anti-GAPDH (Santa Cruz).

Construction and Identification of HER1 and HER2 Double Knockout Cells

In order to knockout the HER1 and HER2 receptors in the genome of HEK293cells, the CRISPR knockout constructs designed specifically for humanHER1 and HER2 were obtained from Santa Cruz Biotechnology. Theconstructs containing gRNA sequences direct the Cas9 protein to induce asite-specific double strand break in the HER1 and HER2 genomic region,which results in the deletion after repairing. To examine thedeletion,primers were designed flanking the gRNA targeting region. Theseprimers were used to perform genomic PCR to reveal the genomic status.

HER1 KO F1: TTTCTTCCAGTTTGCCAAGG (SEQ ID NO:27)

HER1 KO R2: ACGATTCCTGCTCAGCTTGT (SEQ ID NO:28)

HER2 KO F1: GCAAAGGGTTTGAGTGAAGG (SEQ ID NO:29)

HER2 KO R2: GCCACTATGGGAGAAAGGTG (SEQ ID NO:30)

In order to generate stable knockout HER2 cells, the HER2 CRISPRconstructs were transiently transfected into the HEK293 cells. Thetransfected cells were singly plated in 96-well plate 24 hourspost-transfection. Sngle cell clones were selected and expanded. GenomicDNA was prepared. PCR was performed using the primers flanking thetargeted area. To further confirm the HER2 knockout status of the singlecell clones, cellular extract was prepared. Immunoblot was performedusing anti-Her2 antibody. Anti-GAPDH was used as a loading control.

In order to generate the double knockout cells of HER1 and HER2, thesame procedure to knockout HER1 receptor was repeated in the validatedHER2 KO clone. To do so, the CRISPR knockout constructs designedspecifically for human HER1 were obtained from Santa Cruz Biotechnology.The constructs containing gRNA sequences direct the Cas9 protein toinduce a site-specific double strand break in the HER1 genomic region,which results in the deletion after DNA repairing. The CRISPR constructswere transiently transfected into the clone #1 cells. The transfectedcells were singly plated in 96-well plate 24 hours post-transfection. Wescreen 100 single cell clones by genomic PCR using the primers flankingthe targeted area. The deletion of HER1 receptor was further confirmedby the immunoblot assay.

What is claimed is:
 1. A method of determining the activity of a HER2variant, comprising the steps of: a) preparing a cDNA encoding said HER2variant; b) preparing a linear expression cassette containing the HER2variant cDNA; c) transfecting said prepared expression cassettecontaining said HER2 variant cDNA in an assay cell having a JNK reporterconstruct comprising a reporter gene cDNA linked to at least one AP-1binding site, and said cell is capable of expressing HER3; d) exposingsaid transfected cell to a HER3 ligand, wherein said HER3 complexes withsaid HER2 variant to form a dimer which thereby activates the JNK signalconstruct and generates a signal; e) determining the signal activity,wherein a change in signal activity relative to wild type HER2 isindicative of a change in activity of said HER2 variant.
 2. The methodof claim 1, wherein said HER2 variant contains a missense mutation,insertion, or deletion.
 3. The method of claim 1 wherein the expressioncassette containing the HER2 variant cDNA is a linear expressioncassette.
 4. The method of claim 1 wherein said assay cell has a doubleknockout of HER 1 and HER2.
 5. The method of claim 1 wherein the JNKreporter construct comprises a reporter gene cDNA linked to 3 to 12 AP-1binding sites.
 6. The method of claim 1 wherein the JNK reporterconstruct comprises a reporter gene cDNA linked to 6 AP-1 binding sites.7. The method of claim 1 wherein said assay cell further comprises aHER3 expression construct.
 8. The method of claim 7 wherein said HER3expression construct is stably transfected.
 9. The method of claim 1wherein said JNK reporter construct is stably transfected.
 10. Themethod of claim 1, wherein step c) and step d) are performedsimultaneously.
 11. The method of claim 1 wherein steps a) through f)are performed in 32 hours or less.
 12. The method of claim 1, whereinsaid HER2 inhibitor is Lapatinib, Trastuzumab, or Pertuzumab.
 13. Themethod of claim 1, wherein said reporter gene cDNA encodes luciferase,said signal is light emission produced upon addition of a substrate forsaid luciferase, and a decrease in light emission relative to a controlwithout HER2 inhibitor is indicative of the variant being sensitive tothe inhibitor.
 14. The method of claim 1 wherein said HER2 variant isobtained from a patient’s biological sample consisting of blood, serum,and tumor tissue.
 15. The method of claim 1, wherein said HER2 variantis transiently transfected.
 16. The method of claim 1 wherein the JNKreporter construct is SEQ ID NO:,.
 17. A method of determining theactivity of a HER2 variant, comprising the steps of: a) preparing a cDNAencoding said HER2 variant; b) preparing a linear expression cassettecontaining the HER2 variant cDNA; c) transfecting said preparedexpression cassette containing said HER2 variant cDNA in an assay cellhaving a stably integrated JNK reporter construct comprising a reportergene cDNA linked to 6 AP-1 binding sites, said assay cell having astably integrated HER3 expression construct; and said assay cell havinga HER1 and HER2 double knockout; d) exposing said transfected cell to aHER3 ligand, wherein said HER3 complexes with said HER2 variant to forma dimer which thereby activates the JNK signal construct and generates asignal; e) determining the signal activity, wherein a change in signalactivity relative to wild type HER2 is indicative of a change inactivity of said HER2 variant.
 18. An assay cell comprising: a) a stablyintegrated HER3 expression construct; b) a double knockout of HER1 andHER2; and c) a stably integrated JNK signal expression constructcomprising a reporter gene cDNA linked to at least one AP-1 sequence.19. The assay cell of claim 18 wherein said cell is transientlytransfected with a HER2 gene.
 20. The assay cell of claim 18 wherein theparent cell is a HEK293 cell.